Growth differentiation factor-10

ABSTRACT

Growth differentiation factor-10 (GDF-10) is disclosed along with its polynucleotide and amino acid sequence. Also disclosed are diagnostic and therapeutic methods of using the GDF-10 polypeptide and polynucleotide sequences.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The invention relates generally to growth factors andspecifically to a new member of the transforming growth factor beta(TGF-β) super-family, which is denoted, growth differentiation factor-10(GDF-10).

[0003] 2. Description of Related Art

[0004] The transforming growth factor β (TGF-β) superfamily encompassesa group of structurally-related proteins which affect a wide range ofdifferentiation processes during embryonic development. The familyincludes, Mullerian inhibiting substance (MIS), which is required fornormal male sex development (Behringer, et al., Nature, 345:167, 1990),Drosophila decapentaplegic (DPP) gene product, which is required fordorsal-ventral axis formation and morphogenesis of the imaginal disks(Padgett, et al., Nature, 325:81-84, 1987), the Xenopus Vg-1 geneproduct, which localizes to the vegetal pole of eggs ((Weeks, et al.,Cell 51:861-867, 1987), the activins (Mason, et al., Biochem, Biophys.Res. Commun., 135:957-964, 1986), which can induce the formation ofmesoderm and anterior structures in Xenopus embryos (Thomsen, et al.,Cell, 63:485, 1990), and the bone morphogenetic proteins (BMPs,osteogenin, OP-1) which can induce de novo cartilage and bone formation(Sampath, et al., J. Biol. Chem., 265:13198, 1990). The TGF-βs caninfluence a variety of differentiation processes, includingadipogenesis, myogenesis, chondrogenesis, hematopoiesis, and epithelialcell differentiation (for review, see Massague, Cell 49:437, 1987).

[0005] The proteins of the TGF-β family are initially synthesized as alarge precursor protein which subsequently undergoes proteolyticcleavage at a cluster of basic residues approximately 110-140 aminoacids from the C-terminus. The C-terminal regions, or mature regions, ofthe proteins are all structurally related and the different familymembers can be classified into distinct subgroups based on the extent oftheir homology. Although the homologies within particular subgroupsrange from 70% to 90% amino acid sequence identity, the homologiesbetween subgroups are significantly lower, generally ranging from only20% to 50%. In each case, the active species appears to be adisulfide-linked dimer of C-terminal fragments. For most of the familymembers that have been studied, the homodimeric species has been foundto be biologically active, but for other family members, like theinhibins (Ling, et al., Nature, 321:779, 1986) and the TGF-βs (Cheifetz,et al., Cell, 48:409, 1987), heterodimers have also been detected, andthese appear to have different biological properties than the respectivehomodimers.

[0006] Identification of new factors that are tissue-specific in theirexpression pattern will provide a greater understanding of that tissue'sdevelopment and function and allow development of effective diagnosticand therapeutic regimens.

SUMMARY OF THE INVENTION

[0007] The present invention provides a cell growth and differentiationfactor, GDF-10, a polynucleotide sequence which encodes the factor, andantibodies which are immunoreactive with the factor. This factor appearsto relate to various cell proliferative disorders, especially thoseinvolving those involving uterine, nerve, bone, and adipose tissue.

[0008] Thus, in one embodiment, the invention provides a method fordetecting a cell proliferative disorder of uterine, nerve, or fat originand which is associated with GDF-10. In another embodiment, theinvention provides a method for treating a cell proliferative disorderby suppressing or enhancing GDF-10 activity.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009]FIG. 1 shows expression of GDF-10 mRNA in adult tissues.

[0010]FIG. 2 shows nucleotide and predicted amino acid sequence murineGDF-10. Consensus N-glycosylation signals are denoted by plain boxes.

[0011]FIG. 3 shows the alignment of the C-terminal sequences of GDF-10with other members of the TGF-β superfamily. The conserved cysteineresidues are boxed. Dashes denote gaps introduced in order to maximizealignment.

[0012]FIG. 4 shows amino acid homologies with different members of theTGF-β superfamily. Numbers represent percent amino acid identitiesbetween each pair calculated from the first conserved cysteine to theC-terminus.

[0013]FIG. 5 shows an alignment of the C-terminal sequences of human(top lines) and murine (bottom lines) GDF-10.

[0014]FIG. 6 shows an autoradiogram of labeled secreted proteinssynthesized by 293 cells transfected with a pcDNAI vector into which theGDF-10 cDNA was inserted in either the antisense (lanes 1 and 2) orsense (lanes 3 and 4) orientation.

DETAILED DESCRIPTION OF THE INVENTION

[0015] The present invention provides a growth and differentiationfactor, GDF-10 and a polynucleotide sequence encoding GDF-10. GDF-10 isexpressed at highest levels in uterus and fat and at lower levels inother tissues, such as brain. In one embodiment, the invention providesa method for detection of a cell proliferative disorder of uterine,nerve, or fat origin which is associated with GDF-10 expression. Inanother embodiment, the invention provides a method for treating a cellproliferative disorder by using an agent which suppresses or enhancesGDF-10 activity.

[0016] The TGF-β superfamily consists of multifunctional polypeptidesthat control proliferation, differentiation, and other functions in manycell types. Many of the peptides have regulatory, both positive andnegative, effects on other peptide growth factors. The structuralhomology between the GDF-10 protein of this invention and the members ofthe TGF-β family, indicates that GDF-10 is a new member of the family ofgrowth and differentiation factors. Based on the known activities ofmany of the other members, it can be expected that GDF-10 will alsopossess biological activities that will make it useful as a diagnosticand therapeutic reagent.

[0017] The expression of GDF-10 in uterine and fat tissue suggests avariety of applications using the polypeptide, polynucleotide, andantibodies of the invention, related to contraception, fertility,pregnancy, and cell proliferative diseases. Abnormally low levels of thefactor my be indicative of impaired function in the uterus whileabnormally high levels may be indicative of hypertrophy, hyperplasia, orthe presence of ectopic tissue. Hence, GDF-10 my be useful in detectingnot only primary and metastatic neoplasms of uterine origin but indetecting diseases such as endometriosis as well. In addition, GDF-10may also be useful as an indicator of developmental anomalies inprenatal screening procedures.

[0018] Several members of the TGF-β superfamily possess activitiessuggesting possible applications for the treatment of cell proliferativedisorders, such as cancer. In particular, TGF-β has been shown to bepotent growth inhibitor for a variety of cell types (Massague, Cell49:437, 1987). MIS has been shown to inhibit the growth of humanendometrial carcinoma tumors in nude mice (Donahoe, et al., Ann. Surg.194:472, 1981), and inhibin a has been shown to suppress the developmentof tumors both in the ovary and in the testis (Matzuk, et al., Nature,360:313, 1992). GDF-10 may have similar activity and may therefore beuseful as an anti-proliferative agent, such as for the treatment ofendometrial cancer or endometriosis.

[0019] Many of the members of the TGF-β family are also importantmediators of tissue repair. TGF-β has been shown to have marked effectson the formation of collagen and causes of striking angiogenic responsein the newborn mouse (Roberts, et al., Proc. Nat'l Acad. Sci., USA83:4167, 1986). The BMP's can induce new bone growth and are effectivefor the treatment of fractures and other skeletal defects (Glowacki, etal., Lancet, 1:959, 1981; Ferguson, et al., Clin. Orthoped. Relat Res.,227:265, 1988; Johnson, et al., Clin Orthoped Relat. Res., 230:257,1988). Based on the high degree of homology between GDF-10 and BMP-3,GDF-10 may have similar activities and may be useful in repair of tissueinjury caused by trauma or burns for example.

[0020] GDF-10 may play a role in regulation of the menstrual cycle orregulation of uterine function during pregnancy, and therefore, GDF-10,anti-GDF-10 antibodies, or antisense polynucleotides may be usefuleither in contraceptive regimens, in enhancing the success of in vitrofertilization procedures, or in preventing premature labor.

[0021] Certain members of this superfamily have expression patterns orpossess activities that relate to the function of the nervous system.For example, one family member, namely GDNF, has been shown to be apotent neurotrophic factor that can promote the survival of dopaminergicneurons (Lin, et al., Science, 260:1130). Another family member, namelydorsalin, is capable of promoting the differentiation of neural crestcells (Baster, et al., Cell, 73:687). The inhibins and activins havebeen shown to be expressed in the brain (Meunier, et al., Proc. Nat'lAcad. Sci., USA, 85:247, 1988; Sawchenko, et al., Nature, 334:615,1988), and activin has been shown to be capable of functioning as anerve cell survival molecule (Schubert, et al., Nature, 344:868, 1990).Another family member, namely GDF-1, is nervous system-specific in itsexpression pattern (Lee, Proc. Nat'l Acad. Sci., USA, 88:4250, 1991),and certain other family members, such as Vgr-1 (Lyons, et al., Proc.Nat'l Acad. Sci., USA, 86:4554, 1989; Jones et al., Development,111:581, 1991), OP-1 (Ozkaynak, et al., J. Biol. Chem., 267:25220,1992), and BMP-4 (Jones, et al., Development, 111:531, 1991), are alsoknown to be expressed in the nervous system. By analogy GDF-10 may haveapplications in the treatment of neurodegenerative diseases or inmaintaining cells or tissues in culture prior to transplantation.

[0022] The expression of GDF-10 in adipose tissue also raises thepossibility of applications for GDF-10 in the treatment of obesity or ofdisorders related to abnormal proliferation of adipocytes. In thisregard, TGF-β has been shown to be a potent inhibitor of adipocytedifferentiation in vitro (Ignotz and Massague, Proc. Natl. Acad. Sci.,USA 82:8530,1985).

[0023] The term “substantially pure” as used herein refers to GDF-10which is substantially free of other proteins, lipids, carbohydrates orother materials with which it is naturally associated. One skilled inthe art can purify GDF-10 using standard techniques for proteinpurification. The substantially pure polypeptide will yield a singlemajor band on a non-reducing polyacrylamide gel. The purity of theGDF-10 polypeptide can also be determined by amino-terminal amino acidsequence analysis. GDF-10 polypeptide includes functional fragments ofthe polypeptide, as long as the activity of GDF-10 remains. Smallerpeptides containing the biological activity of GDF-10 are included inthe invention.

[0024] The invention provides polynucleotides encoding the GDF-10protein. These polynucleotides include DNA, cDNA and RNA sequences whichencode GDF-10. It is understood that all polynucleotides encoding all ora portion of GDF-10 are also included herein, as long as they encode apolypeptide with GDF-10 activity. Such polynucleotides include naturallyoccurring, synthetic, and intentionally manipulated polynucleotides. Forexample, GDF-10 polynucleotide may be subjected to site-directedmutagenesis. The polynucleotide sequence for GDF-10 also includesantisense sequences. The polynucleotides of the invention includesequences that are degenerate as a result of the genetic code. There are20 natural amino acids, most of which are specified by more than onecodon. Therefore, all degenerate nucleotide sequences are included inthe invention as long as the amino acid sequence of GDF-10 polypeptideencoded by the nucleotide sequence is functionally unchanged.

[0025] Specifically disclosed herein is a cDNA sequence for GDF-10 whichis 2322 base pairs in length and contains an open reading framebeginning with a methionine codon at nucleotide 126. The encodedpolypeptide is 476 amino acids in length with a molecular weight ofabout 52.5 kD, as determined by nucleotide sequence analysis. The GDF-10sequence contains a core of hydrophobic amino acids near the N-terminus,suggestive of a signal sequence for secretion. GDF-10 contains fourpotential N-glycosylation sites at asparagine residues 114, 152, 277,and 467. GDF-10 contains several potential proteolytic processing sites.Cleavage most likely occurs following arginine 365, which would generatea mature fragment of GDF-10 predicted to be 111 amino acids in lengthand have an unglycosylated molecular weight of about 12.6 kD, asdetermined by nucleotide sequence analysis. One skilled in the art canmodify, or partially or completely remove, the glycosyl groups from theGDF-10 protein using standard techniques. Therefore the functionalprotein or fragments thereof of the invention includes glycosylated,partially glycosylated and unglycosylated species of GDF-10.

[0026] The C-terminal region of GDF-10 following the putativeproteolytic processing site shows significant homology to the knownmembers of the TGF-β superfamily. The GDF-10 sequence contains most ofthe residues that are highly conserved in other family members. Amongthe known family mammalian TGF-β family members, GDF-10 is mosthomologous to BMP-3 (83% sequence identity beginning with the firstconserved cysteine residue). GDF-10 also shows significant homology toBMP-3 (approximately 30% sequence identity) in the pro-region of themolecule. Based on these sequence comparisons, GDF-10 and BMP-3 appearto define a new subfamily within the larger superfamily.

[0027] Minor modifications of the recombinant GDF-10 primary amino acidsequence may result in proteins which have substantially equivalentactivity as compared to the GDF-10 polypeptide described herein. Suchmodifications may be deliberate, as by site-directed mutagenesis, or maybe spontaneous. All of the polypeptides produced by these modificationsare included herein as long as the biological activity of GDF-10 stillexists. Further, deletion of one or more amino acids can also result ina modification of the structure of the resultant molecule withoutsignificantly altering its biological activity. This can lead to thedevelopment of a smaller active molecule which would have broaderutility. For example, one can remove amino or carboxy terminal aminoacids which are not required for GDF-10 biological activity.

[0028] The nucleotide sequence encoding the GDF-10 polypeptide of theinvention includes the disclosed sequence and conservative variationsthereof. The term “conservative variation” as used herein denotes thereplacement of an amino acid residue by another, biologically similarresidue. Examples of conservative variations include the substitution ofone hydrophobic residue such as isoleucine, valine, leucine ormethionine for another, or the substitution of one polar residue foranother, such as the substitution of arginine for lysine, glutamic foraspartic acid, or glutamine for asparagine, and the like. The term“conservative variation” also includes the use of a substituted aminoacid in place of an unsubstituted parent amino acid provided thatantibodies raised to the substituted polypeptide also immunoreact withthe unsubstituted polypeptide.

[0029] DNA sequences of the invention can be obtained by severalmethods. For example, the DNA can be isolated using hybridizationtechniques which are well known in the art. These include, but are notlimited to: 1) hybridization of genomic or cDNA libraries with probes todetect homologous nucleotide sequences, 2) polymerase chain reaction(PCR) on genomic DNA or cDNA using primers capable of annealing to theDNA sequence of interest, and 3) antibody screening of expressionlibraries to detect cloned DNA fragments with shared structuralfeatures.

[0030] Preferably the GDF-10 polynucleotide of the invention is derivedfrom a mammalian organism, and most preferably from a mouse, rat, orhuman. Screening procedures which rely on nucleic acid hybridizationmake it possible to isolate any gene sequence from any organism,provided the appropriate probe is available. Oligonucleotide probes,which correspond to a part of the sequence encoding the protein inquestion, can be synthesized chemically. This requires that short,oligopeptide stretches of amino acid sequence must be known. The DNAsequence encoding the protein can be deduced from the genetic code,however, the degeneracy of the code must be taken into account. It ispossible to perform a mixed addition reaction when the sequence isdegenerate. This includes a heterogeneous mixture of denatureddouble-stranded DNA. For such screening, hybridization is preferablyperformed on either single-stranded DNA or denatured double-strandedDNA. Hybridization is particularly useful in the detection of cDNAclones derived from sources where an extremely low amount of mRNAsequences relating to the polypeptide of interest are present. In otherwords, by using stringent hybridization conditions directed to avoidnon-specific binding, it is possible, for example, to allow theautoradiographic visualization of a specific cDNA clone by thehybridization of the target DNA to that single probe in the mixturewhich is its complete complement (Wallace, et al., Nucl. Acid Res.,9:879, 1981).

[0031] The development of specific DNA sequences encoding GDF-10 canalso be obtained by: 1) isolation of double-stranded DNA sequences fromthe genomic DNA; 2) chemical manufacture of a DNA sequence to providethe necessary codons for the polypeptide of interest; and 3) in vitrosynthesis of a double-stranded DNA sequence by reverse transcription ofmRNA isolated from a eukaryotic donor cell; In the latter case, adouble-stranded DNA complement of mRNA is eventually formed which isgenerally referred to as cDNA.

[0032] Of the three above-noted methods for developing specific DNAsequences for use in recombinant procedures, the isolation of genomicDNA isolates is the least common. This is especially true when it isdesirable to obtain the microbial expression of mammalian polypeptidesdue to the presence of introns.

[0033] The synthesis of DNA sequences is frequently the method of choicewhen the entire sequence of amino acid residues of the desiredpolypeptide product is known. When the entire sequence of amino acidresidues of the desired polypeptide is not known, the direct synthesisof DNA sequences is not possible and the method of choice is thesynthesis of cDNA sequences. Among the standard procedures for isolatingcDNA sequences of interest is the formation of plasmid- orphage-carrying cDNA libraries which are derived from reversetranscription of mRNA which is abundant in donor cells that have a highlevel of genetic expression. When used in combination with polymerasechain reaction technology, even rare expression products can be cloned.In those cases where significant portions of the amino acid sequence ofthe polypeptide are known, the production of labeled single ordouble-stranded DNA or RNA probe sequences duplicating a sequenceputatively present in the target cDNA may be employed in DNA/DNAhybridization procedures which are carried out on cloned copies of thecDNA which have been denatured into a single-stranded form (Jay, et al.,Nucl. Acid Res., 11:2325, 1983).

[0034] A cDNA expression library, such as lambda gt11, can be screenedindirectly for GDF-10 peptides having at least one epitope, usingantibodies specific for GDF-10. Such antibodies can be eitherpolyclonally or monoclonally derived and used to detect expressionproduct indicative of the presence of GDF-10 cDNA.

[0035] DNA sequences encoding GDF-10 can be expressed in vitro by DNAtransfer into a suitable host cell. “Host cells” are cells in which avector can be propagated and its DNA expressed. The term also includesany progeny of the subject host cell. It is understood that all progenymay not be identical to the parental cell since there may be mutationsthat occur during replication. However, such progeny are included whenthe term “host cell” is used. Methods of stable transfer, meaning thatthe foreign DNA is continuously maintained in the host, are known in theart.

[0036] In the present invention, the GDF-10 polynucleotide sequences maybe inserted into a recombinant expression vector. The term “recombinantexpression vector” refers to a plasmid, virus or other vehicle known inthe art that has been manipulated by insertion or incorporation of theGDF-10 genetic sequences. Such expression vectors contain a promotersequence which facilitates the efficient transcription of the insertedgenetic sequence of the host. The expression vector typically containsan origin of replication, a promoter, as well as specific genes whichallow phenotypic selection of the transformed cells. Vectors suitablefor use in the present invention include, but are not limited to theT7-based expression vector for expression in bacteria (Rosenberg, etal., Gene, 56:125, 1987), the pMSXND expression vector for expression inmammalian cells (Lee and Nathans, J. Biol. Chem., 263:3521, 1988) andbaculovirus-derived vectors for expression in insect cells. The DNAsegment can be present in the vector operably linked to regulatoryelements, for example, a promoter (e.g., T7, metallothionein I, orpolyhedrin promoters).

[0037] Polynucleotide sequences encoding GDF-10 can be expressed ineither prokaryotes or eukaryotes. Hosts can include microbial, yeast,insect and mammalian organisms. Methods of expressing DNA sequenceshaving eukaryotic or viral sequences in prokaryotes are well known inthe art. Biologically functional viral and plasmid DNA vectors capableof expression and replication in a host are known in the art. Suchvectors are used to incorporate DNA sequences of the invention.Preferably, the mature C-terminal region of GDF-10 is expressed from acDNA clone containing the entire coding sequence of GDF-10.Alternatively, the C-terminal portion of GDF-10 can be expressed as afusion protein with the pro-region of another member of the TGF-β familyor co-expressed with another pro-region (see for example, Hammonds, etal., Molec. Endocrin. 5:149, 1991; Gray, A., and Mason, A., Science,247:1328, 1990).

[0038] Transformation of a host cell with recombinant DNA may be carriedout by conventional techniques as are well known to those skilled in theart. Where the host is prokaryotic, such as E. coli, competent cellswhich are capable of DNA uptake can be prepared from cells harvestedafter exponential growth phase and subsequently treated by the CaCl₂method using procedures well known in the art. Alternatively, MgCl₂ orRbCl can be used. Transformation can also be performed after forming aprotoplast of the host cell if desired.

[0039] When the host is a eukaryote, such methods of transfection of DNAas calcium phosphate co-precipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors may be used. Eukaryotic cells can also becotransformed with DNA sequences encoding the GDF-10 of the invention,and a second foreign DNA molecule encoding a selectable phenotype, suchas the herpes simplex thymidine kinase gene. Another method is to use aeukaryotic viral vector, such as simian virus 40 (SV40) or bovinepapilloma virus, to transiently infect or transform eukaryotic cells andexpress the protein. (see for example, Eukaryotic Viral Vectors, ColdSpring Harbor Laboratory, Gluzman ed., 1982).

[0040] Isolation and purification of microbial expressed polypeptide, orfragments thereof, provided by the invention, may be carried out byconventional means including preparative chromatography andimmunological separations involving monoclonal or polyclonal antibodies.

[0041] The invention includes antibodies immunoreactive with GDF-10polypeptide or functional fragments thereof. Antibody which consistsessentially of pooled monoclonal antibodies with different epitopicspecificities, as well as distinct monoclonal antibody preparations areprovided. Monoclonal antibodies are made from antigen containingfragments of the protein by methods well known to those skilled in theart (Kohler, et al., Nature, 256:495, 1975). The term antibody as usedin this invention is meant to include intact molecules as well asfragments thereof, such as Fab and F(ab′)₂, which are capable of bindingan epitopic determinant on GDF-10.

[0042] The term “cell-proliferative disorder” denotes malignant as wellas non-malignant cell populations which often appear to differ from thesurrounding tissue both morphologically and genotypically. The term“cell-proliferative disorder” also includes situations in which anormally occurring process could be enhanced or suppressed for clinicalbenefit; an example of such a process would be fracture healing.Malignant cells (i.e. cancer) develop as a result of a multistepprocess. The GDF-10 polynucleotide that is an antisense molecule isuseful in treating malignancies of the various organ systems,particularly, for example, cells in uterine or adipose tissue.Essentially, any disorder which is etiologically linked to alteredexpression of GDF-10 could be considered susceptible to treatment with aGDF-10 suppressing reagent. One such disorder is a malignant cellproliferative disorder, for example.

[0043] The invention provides a method for detecting a cellproliferative disorder of uterine or adipose tissue which comprisescontacting an anti-GDF-10 antibody with a cell suspected of having aGDF-10 associated disorder and detecting binding to the antibody. Theantibody reactive with GDF-10 is labeled with a compound which allowsdetection of binding to GDF-10. For purposes of the invention, anantibody specific for GDF-10 polypeptide may be used to detect the levelof GDF-10 in biological fluids and tissues. Any specimen containing adetectable amount of antigen can be used. A preferred sample of thisinvention is uterine or fat tissue. The level of GDF-10 in the suspectcell can be compared with the level in a normal cell to determinewhether the subject has a GDF-10-associated cell proliferative disorder.Preferably the subject is human.

[0044] The antibodies of the invention can be used in any subject inwhich it is desirable to administer in vitro or in vivo immunodiagnosisor immunotherapy. The antibodies of the invention are suited for use,for example, in immunoassays in which they can be utilized in liquidphase or bound to a solid phase carrier. In addition, the antibodies inthese immunoassays can be detectably labeled in various ways. Examplesof types of immunoassays which can utilize antibodies of the inventionare competitive and non-competitive immunoassays in either a direct orindirect format. Examples of such immunoassays are the radioimmunoassay(RIA) and the sandwich (immunometric) assay. Detection of the antigensusing the antibodies of the invention can be done utilizing immunoassayswhich are run in either the forward, reverse, or simultaneous modes,including immunohistochemical assays on physiological samples. Those ofskill in the art will know, or can readily discern, other immunoassayformats without undue experimentation.

[0045] The antibodies of the invention can be bound to many differentcarriers and used to detect the presence of an antigen comprising thepolypeptide of the invention. Examples of well-known carriers includeglass, polystyrene, polypropylene, polyethylene, dextran, nylon,amylases, natural and modified celluloses, polyacrylamides, agaroses andmagnetite. The nature of the carrier can be either soluble or insolublefor purposes of the invention. Those skilled in the art will know ofother suitable carriers for binding antibodies, or will be able toascertain such, using routine experimentation.

[0046] There are many different labels and methods of labeling known tothose of ordinary skill in the art. Examples of the types of labelswhich can be used in the present invention include enzymes,radioisotopes, fluorescent compounds, colloidal metals, chemiluminescentcompounds, phosphorescent compounds, and bioluminescent compounds. Thoseof ordinary skill in the art will know of other suitable labels forbinding to the antibody, or will be able to ascertain such, usingroutine experimentation.

[0047] Another technique which may also result in greater sensitivityconsists of coupling the antibodies to low molecular weight haptens.These haptens can then be specifically detected by means of a secondreaction. For example, it is common to use such haptens as biotin, whichreacts with avidin, or dinitrophenyl, puridoxal, and fluorescein, whichcan react with specific antihapten antibodies.

[0048] In using the monoclonal antibodies of the invention for the invivo detection of antigen, the detectably labeled antibody is given adose which is diagnostically effective. The term “diagnosticallyeffective” means that the amount of detectably labeled monoclonalantibody is administered in sufficient quantity to enable detection ofthe site having the antigen comprising a polypeptide of the inventionfor which the monoclonal antibodies are specific.

[0049] The concentration of detectably labeled monoclonal antibody whichis administered should be sufficient such that the binding to thosecells having the polypeptide is detectable compared to the background.Further, it is desirable that the detectably labeled monoclonal antibodybe rapidly cleared from the circulatory system in order to give the besttarget-to-background signal ratio.

[0050] As a rule, the dosage of detectably labeled monoclonal antibodyfor in vivo diagnosis will vary depending on such factors as age, sex,and extent of disease of the individual. Such dosages may vary, forexample, depending on whether multiple injections are given, antigenicburden, and other factors known to those of skill in the art.

[0051] For in vivo diagnostic imaging, the type of detection instrumentavailable is a major factor in selecting a given radioisotope. Theradioisotope chosen must have a type of decay which is detectable for agiven type of instrument. Still another important factor in selecting aradioisotope for in vivo diagnosis is that deleterious radiation withrespect to the host is minimized. Ideally, a radioisotope used for invivo imaging will lack a particle emission, but produce a large numberof photons in the 140-250 keV range, which may readily be detected byconventional gamma cameras.

[0052] For in vivo diagnosis radioisotopes may be bound toimmunoglobulin either directly or indirectly by using an intermediatefunctional group. Intermediate functional groups which often are used tobind radioisotopes which exist as metallic ions to immunoglobulins arethe bifunctional chelating agents such as diethylenetriaminepentaceticacid (DTPA) and ethylenediaminetetraacetic acid (EDTA) and similarmolecules. Typical examples of metallic ions which can be bound to themonoclonal antibodies of the invention are ¹¹¹In, ⁹⁷Ru, ⁶⁷Ga, ⁶⁸Ga,⁷²As, ⁸⁹Zr, and ²⁰¹Tl.

[0053] The monoclonal antibodies of the invention can also be labeledwith a paramagnetic isotope for purposes of in vivo diagnosis, as inmagnetic resonance imaging (MRI) or electron spin resonance (ESR). Ingeneral, any conventional method for visualizing diagnostic imaging canbe utilized. Usually gamma and positron emitting radioisotopes are usedfor camera imaging and paramagnetic isotopes for MRI. Elements which areparticularly useful in such techniques include ¹⁵⁷Gd, ⁵⁵Mn, ¹⁶²Dy, ⁵²Cr,and ⁵⁶Fe.

[0054] The monoclonal antibodies of the invention can be used in vitroand in vivo to monitor the course of amelioration of a GDF-10-associateddisease in a subject. Thus, for example, by measuring the increase ordecrease in the number of cells expressing antigen comprising apolypeptide of the invention or changes in the concentration of suchantigen present in various body fluids, it would be possible todetermine whether a particular therapeutic regimen aimed at amelioratingthe GDF-10-associated disease is effective. The term “ameliorate”denotes a lessening of the detrimental effect of the GDF-10-associateddisease in the subject receiving therapy.

[0055] The present invention identifies a nucleotide sequence that canbe expressed in an altered manner as compared to expression in a normalcell, therefore it is possible to design appropriate therapeutic ordiagnostic techniques directed to this sequence. Thus, where acell-proliferative disorder is associated with the expression of GDF-10,nucleic acid sequences that interfere with GDF-10 expression at thetranslational level can be used. This approach utilizes, for example,antisense nucleic acid and ribozymes to block translation of a specificGDF-10 mRNA, either by masking that mRNA with an antisense nucleic acidor by cleaving it with a ribozyme.

[0056] Antisense nucleic acids are DNA or RNA molecules that arecomplementary to at least a portion of a specific mRNA molecule(Weintraub, Scientific American, 262:40, 1990). In the cell, theantisense nucleic acids hybridize to the corresponding mRNA, forming adouble-stranded molecule. The antisense nucleic acids interfere with thetranslation of the mRNA, since the cell will not translate a mRNA thatis double-stranded. Antisense oligomers of about 15 nucleotides arepreferred, since they are easily synthesized and are less likely tocause problems than larger molecules when introduced into the targetGDF-10-producing cell. The use of antisense methods to inhibit the invitro translation of genes is well known in the art (Marcus-Sakura,Anal. Biochem., 172:289, 1988).

[0057] Ribozymes are RNA molecules possessing the ability tospecifically cleave other single-stranded RNA in a manner analogous toDNA restriction endonucleases. Through the modification of nucleotidesequences which encode these RNAs, it is possible to engineer moleculesthat recognize specific nucleotide sequences in an RNA molecule andcleave it (Cech, J.Amer.Med. Assn., 260:3030, 1988). A major advantageof this approach is that, because they are sequence-specific, only mRNAswith particular sequences are inactivated.

[0058] There are two basic types of ribozymes namely, tetrahymena-type(Hasselhoff, Nature, 334:585, 1988) and “hammerhead”-type.Tetrahymena-type ribozymes recognize sequences which are four bases inlength, while “hammerhead”-type ribozymes recognize base sequences 11-18bases in length. The longer the recognition sequence, the greater thelikelihood that the sequence will occur exclusively in the target mRNAspecies. Consequently, hammerhead-type ribozymes are preferable totetrahymena-type ribozymes for inactivating a specific mRNA species and18-based recognition sequences are preferable to shorter recognitionsequences.

[0059] The present invention also provides gene therapy for thetreatment of cell proliferative disorders which are mediated by GDF-10protein. Such therapy would achieve its therapeutic effect byintroduction of the GDF-10 antisense polynucleotide into cells havingthe proliferative disorder. Delivery of antisense GDF-10 polynucleotidecan be achieved using a recombinant expression vector such as a chimericvirus or a colloidal dispersion system. Especially preferred fortherapeutic delivery of antisense sequences is the use of targetedliposomes.

[0060] Various viral vectors which can be utilized for gene therapy astaught herein include adenovirus, herpes virus, vaccinia, or,preferably, an RNA virus such as a retrovirus. Preferably, theretroviral vector is a derivative of a murine or avian retrovirus.Examples of retroviral vectors in which a single foreign gene can beinserted include, but are not limited to: Moloney murine leukemia virus(MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumorvirus (MuMTV), and Rous Sarcoma Virus (RSV). A number of additionalretroviral vectors can incorporate multiple genes. All of these vectorscan transfer or incorporate a gene for a selectable marker so thattransduced cells can be identified and generated. By inserting a GDF-10sequence of interest into the viral vector, along with another genewhich encodes the ligand for a receptor on a specific target cell, forexample, the vector is now target specific. Retroviral vectors can bemade target specific by inserting, for example, a polynucleotideencoding a sugar, a glycolipid, or a protein. Preferred targeting isaccomplished by using an antibody to target the retroviral vector. Thoseof skill in the art will know of, or can readily ascertain without undueexperimentation, specific polynucleotide sequences which can be insertedinto the retroviral genome to allow target specific delivery of theretroviral vector containing the GDF-10 antisense polynucleotide.

[0061] Since recombinant retroviruses are defective, they requireassistance in order to produce infectious vector particles. Thisassistance can be provided, for example, by using helper cell lines thatcontain plasmids encoding all of the structural genes of the retrovirusunder the control of regulatory sequences within the LTR. These plasmidsare missing a nucleotide sequence which enables the packaging mechanismto recognize an RNA transcript for encapsidation. Helper cell lineswhich have deletions of the packaging signal include, but are notlimited to Ψ2, PA317 and PA12, for example. These cell lines produceempty virions, since no genome is packaged. If a retroviral vector isintroduced into such cells in which the packaging signal is intact, butthe structural genes are replaced by other genes of interest, the vectorcan be packaged and vector virion produced.

[0062] Alternatively, NIH 3T3 or other tissue culture cells can bedirectly transfected with plasmids encoding the retroviral structuralgenes gag, pol and env, by conventional calcium phosphate transfection.These cells are then transfected with the vector plasmid containing thegenes of interest. The resulting cells release the retroviral vectorinto the culture medium.

[0063] Another targeted delivery system for GDF-10 antisensepolynucleotides is a colloidal dispersion system. Colloidal dispersionsystems include macromolecule complexes, nanocapsules, microspheres,beads, and lipid-based systems including oil-in-water emulsions,micelles, mixed micelles, and liposomes. The preferred colloidal systemof this invention is a liposome. Liposomes are artificial membranevesicles which are useful as delivery vehicles in vitro and in vivo. Ithas been shown that large unilamellar vesicles (LUV), which range insize from 0.2-4.0 μm can encapsulate a substantial percentage of anaqueous buffer containing large macromolecules. RNA, DNA and intactvirions can be encapsulated within the aqueous interior and be deliveredto cells in a biologically active form (Fraley, et al., Trends Biochem.Sci., 6:77,1981). In addition to mammalian cells, liposomes have beenused for delivery of polynucleotides in plant, yeast and bacterialcells. In order for a liposome to be an efficient gene transfer vehicle,the following characteristics should be present: (1) encapsulation ofthe genes of interest at high efficiency while not compromising theirbiological activity; (2) preferential and substantial binding to atarget cell in comparison to non-target cells; (3) delivery of theaqueous contents of the vesicle to the target cell cytoplasm at highefficiency; and (4) accurate and effective expression of geneticinformation (Mannino, et al., Biotechniques, 6:682, 1988).

[0064] The composition of the liposome is usually a combination ofphospholipids, particularly high-phase-transition-temperaturephospholipids, usually in combination with steroids, especiallycholesterol. Other phospholipids or other lipids may also be used. Thephysical characteristics of liposomes depend on pH, ionic strength, andthe presence of divalent cations.

[0065] Examples of lipids useful in liposome production includephosphatidyl compounds, such as phosphatidylglycerol,phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine,sphingolipids, cerebrosides, and gangliosides. Particularly useful arediacylphosphatidylglycerols, where the lipid moiety contains from 14-18carbon atoms, particularly from 16-18 carbon atoms, and is saturated.Illustrative phospholipids include egg phosphatidylcholine,dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.

[0066] The targeting of liposomes can be classified based on anatomicaland mechanistic factors. Anatomical classification is based on the levelof selectivity, for example, organ-specific, cell-specific, andorganelle-specific. Mechanistic targeting can be distinguished basedupon whether it is passive or active. Passive targeting utilizes thenatural tendency of liposomes to distribute to cells of thereticulo-endothelial system (RES) in organs which contain sinusoidalcapillaries. Active targeting, on the other hand, involves alteration ofthe liposome by coupling the liposome to a specific ligand such as amonoclonal antibody, sugar, glycolipid, or protein, or by changing thecomposition or size of the liposome in order to achieve targeting toorgans and cell types other than the naturally occurring sites oflocalization.

[0067] The surface of the targeted delivery system may be modified in avariety of ways. In the case of a liposomal targeted delivery system,lipid groups can be incorporated into the lipid bilayer of the liposomein order to maintain the targeting ligand in stable association with theliposomal bilayer. Various linking groups can be used for joining thelipid chains to the targeting ligand.

[0068] Due to the expression of GDF-10 primarily in uterine and adiposetissue, there are a variety of applications using the polypeptide,polynucleotide, and antibodies of the invention, related to these andother tissues. Such applications include treatment of cell proliferativedisorders involving these and other tissues, including bone. Inaddition, GDF-10 may be useful in various gene therapy procedures.

[0069] The following examples are intended to illustrate but not limitthe invention. While they are typical of those that might be used, otherprocedures known to those skilled in the art may alternatively be used.

EXAMPLE 1 Identification and Isolation of a Novel TGF-β Family Member

[0070] To identify new members of the TGF-β superfamily, degenerateoligonucleotides were designed which corresponded to two conservedregions among the known family members: one region downstream of thefirst conserved cysteine residue and the other region spanning theinvariant cysteine residues near the C-terminus. These primers were usedfor polymerase chain reactions on lung and brain cDNA followed bysubcloning the PCR products using restriction sites placed at the 5′ends of the primers, picking individual E. coli colonies carrying thesesubcloned inserts, and using a combination of random sequencing andhybridization analysis to eliminate know members of the superfamily.

[0071] GDF-10 was identified from a mixture of PCR products obtainedwith the primers: NSC1:5′-CCGGAATTCAA(G/A)GT(G/A/T/C)GA(T/C)TT(T/C)GC(G/A/T/C)GA         (T/C)AT(A/C/T)GG(G/A,T/C)TGG-3′ NSC2:5′-CCGGAATTC(A/G)CA(G/A/T/C)GC(A/G)CA(G/A)CT(T/C)TC(G/A/T/C)         AC(G/A/T/C)GTCAT-3′ NSC3:5′-CCGGAATTC(A/G)CA(G/A/T/C)GC(A/G)CA(G/A/T/C)GA(T/C)TC         (G/A/T/C)AC(G/A/T/C)GTCAT-3′

[0072] PCR using primers NSC1 with NSC2 or NSC1 with NSC3 was carriedout with cDNA prepared from 0.25 μg of lung or brain mRNA for 35 cyclesat 94° C. for 1 min, 50° C. for 2 min, and 72° C. for 2 min. PCRproducts of approximately 300 base pairs were digested with Eco RI, gelpurified, and subcloned in the Bluescript vector (Stratagene, San Diego,Calif.). DNA was prepared from bacterial colonies carrying individualsubclones and sequenced. Of 11 clones that were sequenced, 9corresponded to BMP-3, and two represented a novel sequence, which wasdesignated GDF-10.

EXAMPLE 2 Expression Pattern and Sequence of GDF-10

[0073] To determine the expression pattern of GDF-10, RNA samplesprepared from a variety of adult tissues were screened by Northernanalysis. 2.5 micrograms of twice polyA-selected RNA prepared from eachtissue were electrophoresed on formaldehyde gels, blotted and probedwith GDF-10. As shown in FIG. 1, the GDF-10 probe detected an mRNAexpressed at highest levels in uterus, fat, and brain.

[0074] A murine uterus cDNA library consisting of 3×10⁶ recombinantphage was constructed in lambda ZAP II and screened with a probe derivedfrom the GDF-10 PCR product. The entire nucleotide sequence of thelongest of 7 hybridizing clones is shown in FIG. 2. ConsensusN-glycosylation signals are denoted by plain boxes. Numbers indicatenucleotide position relative to the 5′ end. The 2322 bp sequencecontains a long open reading frame beginning with a methionine codon atnucleotide 126 and potentially encoding a protein 476 amino acids inlength with a molecular weight of 52.5 kD. The predicted GDF-10 aminoacid sequence contains a hydrophobic N-terminal region, suggestive of asignal sequence for secretion, four potential N-linked glycosylationsites at asparagine residues 114, 152, 277, and 467 and a putativeproteolytic processing site at amino acid 365. Cleavage of the GDF-10precursor at this site would generate a mature GDF-10 protein 111 aminoacids in length with a predicted unglycosylated molecular weight of 12.6kD.

[0075] The C-terminal region of GDF-10 following the putativeproteolytic processing site shows significant homology to the knownmembers of the TGF-β superfamily (FIG. 3). FIG. 3 shows the alignment ofthe C-terminal sequences of GDF-10 with the corresponding regions ofhuman GDF-1 (Lee, Proc. Natl. Acad. Sci. USA, 88:4250-4254, 1991),murine GDF-3 and GDF-9 (McPherron and Lee, J. Biol. Chem. 268:3444,1993), human BMP-2 and 4 (Wozney, et al., Science, 242:1528-1534, 1988),human Vgr-1 (Celeste, et al., Proc. Natl. Acad. Sci. USA, 87:9843-9847,1990), human OP-1 (Ozkaynak, et al., EMBO J., 9:2085-2093, 1990), humanBMP-5 (Celeste, et al., Proc. Natl. Acad. Sci. USA, 87:9843-9847, 1990),human OP-2 (Ozkaynak, et al., J. Biol. Chem., 267:25220-25227, 1992),human BMP-3 (Wozney, et al., Science, 242:1528-1534, 1988), human MIS(Cate, et al., Cell, 45:685-698, 1986), human inhibin alpha, βA, and βB(Mason, et al., Biochem, Biophys. Res. Commun., 135:957-964, 1986),murine nodal (Zhou, et al., Nature, 361:543-547, 1993), human TGF-β1(Derynck, et al., Nature, 316:701-705, 1985), humanTGF-β2 (deMartin, etal., EMBO J., 6:3673-3677, 1987), and human TGF-β3 (ten Dijke, et al.,Proc. Natl. Acad. Sci. USA 85:47154719,1988). The conserved cysteineresidues are boxed. Dashes denote gaps introduced in order to maximizethe alignment.

[0076] GDF-10 contains most of the residues that are highly conserved inother family members, including the seven cysteine residues with theircharacteristic spacing.

[0077]FIG. 4 shows the amino acid homologies among the different membersof the TGF-β superfamily. Numbers represent percent amino acididentities calculated from the first conserved cysteine to theC-terminus. In this region, GDF-10 is most homologous to BMP-3 (83%sequence identity).

EXAMPLE 3 Isolation of Human GDF-10

[0078] To isolate human GDF-10, a human uterus cDNA library consistingof 16.2×10⁶ recombinant phage was constructed in lambda ZAP II andscreened with a murine GDF-10 probe. From this library, 20 hybridizingclones were isolated. Partial nucleotide sequence analysis of thelongest clone showed that human and murine GDF-10 are highly homologous;the predicted amino acid sequences are 97% identical beginning with thefirst conserved cysteine residue following the predicted cleavage site(FIG. 5).

EXAMPLE 4 Secretion of GDF-10 by Mammalian Cells

[0079] To determine whether GDF-10 is secreted by mammalian cells, theGDF-10 cDNA was cloned into the pcDNAI expression vector and transfectedinto 293 cells. Following DNA transfection, the cells were metabolicallylabeled with a mixture of [³⁵S]-cysteine and [³⁵S]-methionine, andlabeled secreted proteins were analyzed by SDS-polyacrylamide gelelectrophoresis. As shown in FIG. 6, additional bands were detected incells transfected with a sense GDF-10 construct compared to an antisensecontrol construct. The presence of multiple protein species most likelyindicates that 293 cells are capable of proteolytically processingGDF-10. Hence, these data suggest that GDF-10 is secreted by these cellsand that GDF-10 is cleaved, as predicted from the cDNA sequence.

[0080] Although the invention has been described with reference to thepresently preferred embodiment, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims.

1 26 36 base pairs nucleic acid single linear DNA (genomic) NSC1 CDS1..36 1 CCGGAATTCA ARGTNGAYTT YGCNGAYATH GGNTGG 36 33 base pairs nucleicacid single linear DNA (genomic) NSC2 CDS 1..33 2 CCGGAATTCR CANGCRCARCTYTCNACNGT CAT 33 33 base pairs nucleic acid single linear DNA (genomic)NSC3 CDS 1..33 3 CCGGAATTCR CANGCRCANG AYTCNACNGT CAT 33 2322 base pairsnucleic acid single linear DNA (genomic) Murine GDF-10 CDS 126..1553 4TGGGGTCATC CGGGCTGTCC GAGTCCCACA GGGACAACTC CAGCCGCGGA CGAGGTGCAC 60AGCCAACACT GAGCCCTCCT TGTCTGTTCT CCTGGGCTCA GACCCTTCAC CACCGTTACT 120CAGCC ATG GCT CCA GGT CCT GCT CGG ATC AGC TTG GGG TCC CAG CTG 167 MetAla Pro Gly Pro Ala Arg Ile Ser Leu Gly Ser Gln Leu 1 5 10 CTG CCC ATGGTG CCG CTG CTC CTG CTG CTG CGG GGC GCA GGC TGC GGC 215 Leu Pro Met ValPro Leu Leu Leu Leu Leu Arg Gly Ala Gly Cys Gly 15 20 25 30 CAC AGG GGCCCC TCA TGG TCC TCA TTG CCC TCG GCA GCT GCC GGT CTG 263 His Arg Gly ProSer Trp Ser Ser Leu Pro Ser Ala Ala Ala Gly Leu 35 40 45 CAG GGG GAC AGGGAC TCC CAG CAG TCA CCC GGG GAC GCA GCA GCC GCT 311 Gln Gly Asp Arg AspSer Gln Gln Ser Pro Gly Asp Ala Ala Ala Ala 50 55 60 CTG GGC CCA GGC GCCCAG GAC ATG GTC GCT ATC CAC ATG CTC AGG CTC 359 Leu Gly Pro Gly Ala GlnAsp Met Val Ala Ile His Met Leu Arg Leu 65 70 75 TAT GAG AAG TAC AAC CGAAGA GGT GCT CCA CCG GGA GGA GGC AAC ACC 407 Tyr Glu Lys Tyr Asn Arg ArgGly Ala Pro Pro Gly Gly Gly Asn Thr 80 85 90 GTC CGA AGC TTC CGT GCC CGGCTG GAA ATG ATC GAC CAA AAG CCT GTG 455 Val Arg Ser Phe Arg Ala Arg LeuGlu Met Ile Asp Gln Lys Pro Val 95 100 105 110 TAT TTC TTC AAC TTG ACTTCC ATG CAA GAC TCA GAA ATG ATC CTC ACA 503 Tyr Phe Phe Asn Leu Thr SerMet Gln Asp Ser Glu Met Ile Leu Thr 115 120 125 GCC GCC TTC CAC TTC TACTCA GAA CCT CCA CGG TGG CCC CGG GCT GGT 551 Ala Ala Phe His Phe Tyr SerGlu Pro Pro Arg Trp Pro Arg Ala Gly 130 135 140 GAG GTA TTC TGC AAG CCCCGA GCT AAG AAC GCA TCC TGC CGC CTC CTG 599 Glu Val Phe Cys Lys Pro ArgAla Lys Asn Ala Ser Cys Arg Leu Leu 145 150 155 ACC CCA GGG CTG CCT GCACGC TTG CAC CTA ATC TTC CGC AGT CTT TCC 647 Thr Pro Gly Leu Pro Ala ArgLeu His Leu Ile Phe Arg Ser Leu Ser 160 165 170 CAG AAC ACC GCC ACT CAGGGG CTG CTC CGC GGG GCC ATG GCC CTG ACG 695 Gln Asn Thr Ala Thr Gln GlyLeu Leu Arg Gly Ala Met Ala Leu Thr 175 180 185 190 CCT CCA CCA CGT GGCCTG TGG CAG GCC AAG GAC ATC TCC TCA ATC ATC 743 Pro Pro Pro Arg Gly LeuTrp Gln Ala Lys Asp Ile Ser Ser Ile Ile 195 200 205 AAG GCT GCC CGA AGGGAT GGA GAG CTG CTT CTC TCT GCT CAG CTG GAT 791 Lys Ala Ala Arg Arg AspGly Glu Leu Leu Leu Ser Ala Gln Leu Asp 210 215 220 ACT GGG GAG AAG GACCCC GGA GTG CCA CGG CCC AGT TCC CAC ATG CCC 839 Thr Gly Glu Lys Asp ProGly Val Pro Arg Pro Ser Ser His Met Pro 225 230 235 TAT ATC CTT GTC TACGCC AAT GAC CTG GCC ATC TCC GAA CCC AAC AGT 887 Tyr Ile Leu Val Tyr AlaAsn Asp Leu Ala Ile Ser Glu Pro Asn Ser 240 245 250 GTA GCA GTG TCG CTACAG AGA TAC GAC CCA TTT CCA GCT GGA GAC TTT 935 Val Ala Val Ser Leu GlnArg Tyr Asp Pro Phe Pro Ala Gly Asp Phe 255 260 265 270 GAG CCT GGA GCAGCC CCC AAC AGC TCA GCT GAT CCC CGC GTG CGC AGG 983 Glu Pro Gly Ala AlaPro Asn Ser Ser Ala Asp Pro Arg Val Arg Arg 275 280 285 GCG GCT CAG GTGTCA AAA CCC CTG CAA GAC AAT GAA CTG CCG GGG CTG 1031 Ala Ala Gln Val SerLys Pro Leu Gln Asp Asn Glu Leu Pro Gly Leu 290 295 300 GAT GAA AGA CCAGCG CCT GCC CTG CAT GCC CAG AAT TTC CAC AAG CAC 1079 Asp Glu Arg Pro AlaPro Ala Leu His Ala Gln Asn Phe His Lys His 305 310 315 GAG TTC TGG TCCAGT CCT TTC CGG GCA CTG AAA CCC CGC ACG GCG CGC 1127 Glu Phe Trp Ser SerPro Phe Arg Ala Leu Lys Pro Arg Thr Ala Arg 320 325 330 AAA GAC CGC AAGAAG AAG GAC CAG GAC ACA TTC ACC GCC GCC TCC TCT 1175 Lys Asp Arg Lys LysLys Asp Gln Asp Thr Phe Thr Ala Ala Ser Ser 335 340 345 350 CAG GTG CTGGAC TTT GAC GAG AAG ACG ATG CAG AAA GCC AGG AGG CGG 1223 Gln Val Leu AspPhe Asp Glu Lys Thr Met Gln Lys Ala Arg Arg Arg 355 360 365 CAG TGG GATGAG CCC CGG GTC TGC TCC AGG AGG TAC CTG AAG GTG GAT 1271 Gln Trp Asp GluPro Arg Val Cys Ser Arg Arg Tyr Leu Lys Val Asp 370 375 380 TTT GCA GACATC GGG TGG AAT GAA TGG ATC ATC TCT CCC AAA TCC TTT 1319 Phe Ala Asp IleGly Trp Asn Glu Trp Ile Ile Ser Pro Lys Ser Phe 385 390 395 GAC GCC TACTAC TGT GCT GGG GCC TGC GAG TTC CCC ATG CCC AAG ATT 1367 Asp Ala Tyr TyrCys Ala Gly Ala Cys Glu Phe Pro Met Pro Lys Ile 400 405 410 GTC CGC CCATCC AAC CAT GCC ACC ATC CAG AGC ATC GTC AGA GCT GTG 1415 Val Arg Pro SerAsn His Ala Thr Ile Gln Ser Ile Val Arg Ala Val 415 420 425 430 GGC ATTGTC CCT GGC ATC CCA GAG CCA TGC TGT GTT CCA GAC AAG ATG 1463 Gly Ile ValPro Gly Ile Pro Glu Pro Cys Cys Val Pro Asp Lys Met 435 440 445 AAC TCCCTT GGA GTC CTT TTC CTG GAT GAA AAT CGG AAT GCG GTT CTG 1511 Asn Ser LeuGly Val Leu Phe Leu Asp Glu Asn Arg Asn Ala Val Leu 450 455 460 AAG GTGTAC CCC AAT ATG TCC GTA GAG ACC TGT GCC TGT CGG 1553 Lys Val Tyr Pro AsnMet Ser Val Glu Thr Cys Ala Cys Arg 465 470 475 TAAGATGGCT TCAAGATAGAAGACAGACCT GCTTCATCCC TGCCCTGCAG AGTGGCAATC 1613 TTGGAGCCAG GGACTTGACTCGGGGAGGTT CCAGGTGCTA GACAGAGCTT ACAGGCAGCC 1673 CTGCTGGGAC CAAGAAAGATCTGCCCACCA CATCGCAATT CTTCAGTTCT TCCGTGCTGG 1733 TGGTAGCTCT GTAAAGACGTGTTGAGTTCC TGGAAGAAAT CTGGAATTAA CTGTGGTCTG 1793 CAATTTGCCC ATCATCCCTGCCCACACTTT TCAAGGCCTA GAAATAACGT GTGTCCTCAA 1853 ATGTCAACTC CAGGCATTTGTCCTCTCAAA ACCTAGAAAG ACTATGCAAA TCTTGGGGTA 1913 CTCCCCCCCC CCATGGCAGTTTAAATGCTG TTTTAAAACC CTCAGGCTGC ATTCTAGAAA 1973 CAGGGCCTAA CCCATGGCACGAGTGAGTAT TTTCTCTTAC GTTTCACTAC ACGTGCTTTT 2033 ATACATGCAG TATGCACATGTAATCACGGT TGATTTCTTC TTTTAATATA TGTATTTCTA 2093 TTTCAAAGCA AAACGGAGAGAGTCGATCCC ATCCCCTGCA GAGGTAATAA TGCAAGTTAG 2153 GTGTGGGTTG TCTAAGCATGTGTATGGAAA TAATACATAC AGTAATATGC TGGAATACTA 2213 AAAAAGTAAC CAAGATTTTATATTTTTGTA AATTATACTT TGTATACTGT AGATTGTGAG 2273 TGTTCTGTGT TTTTATGGAAAGCTAATAAA TTAAAGGTGC GGAGGTATC 2322 476 amino acids amino acid linearprotein 5 Met Ala Pro Gly Pro Ala Arg Ile Ser Leu Gly Ser Gln Leu LeuPro 1 5 10 15 Met Val Pro Leu Leu Leu Leu Leu Arg Gly Ala Gly Cys GlyHis Arg 20 25 30 Gly Pro Ser Trp Ser Ser Leu Pro Ser Ala Ala Ala Gly LeuGln Gly 35 40 45 Asp Arg Asp Ser Gln Gln Ser Pro Gly Asp Ala Ala Ala AlaLeu Gly 50 55 60 Pro Gly Ala Gln Asp Met Val Ala Ile His Met Leu Arg LeuTyr Glu 65 70 75 80 Lys Tyr Asn Arg Arg Gly Ala Pro Pro Gly Gly Gly AsnThr Val Arg 85 90 95 Ser Phe Arg Ala Arg Leu Glu Met Ile Asp Gln Lys ProVal Tyr Phe 100 105 110 Phe Asn Leu Thr Ser Met Gln Asp Ser Glu Met IleLeu Thr Ala Ala 115 120 125 Phe His Phe Tyr Ser Glu Pro Pro Arg Trp ProArg Ala Gly Glu Val 130 135 140 Phe Cys Lys Pro Arg Ala Lys Asn Ala SerCys Arg Leu Leu Thr Pro 145 150 155 160 Gly Leu Pro Ala Arg Leu His LeuIle Phe Arg Ser Leu Ser Gln Asn 165 170 175 Thr Ala Thr Gln Gly Leu LeuArg Gly Ala Met Ala Leu Thr Pro Pro 180 185 190 Pro Arg Gly Leu Trp GlnAla Lys Asp Ile Ser Ser Ile Ile Lys Ala 195 200 205 Ala Arg Arg Asp GlyGlu Leu Leu Leu Ser Ala Gln Leu Asp Thr Gly 210 215 220 Glu Lys Asp ProGly Val Pro Arg Pro Ser Ser His Met Pro Tyr Ile 225 230 235 240 Leu ValTyr Ala Asn Asp Leu Ala Ile Ser Glu Pro Asn Ser Val Ala 245 250 255 ValSer Leu Gln Arg Tyr Asp Pro Phe Pro Ala Gly Asp Phe Glu Pro 260 265 270Gly Ala Ala Pro Asn Ser Ser Ala Asp Pro Arg Val Arg Arg Ala Ala 275 280285 Gln Val Ser Lys Pro Leu Gln Asp Asn Glu Leu Pro Gly Leu Asp Glu 290295 300 Arg Pro Ala Pro Ala Leu His Ala Gln Asn Phe His Lys His Glu Phe305 310 315 320 Trp Ser Ser Pro Phe Arg Ala Leu Lys Pro Arg Thr Ala ArgLys Asp 325 330 335 Arg Lys Lys Lys Asp Gln Asp Thr Phe Thr Ala Ala SerSer Gln Val 340 345 350 Leu Asp Phe Asp Glu Lys Thr Met Gln Lys Ala ArgArg Arg Gln Trp 355 360 365 Asp Glu Pro Arg Val Cys Ser Arg Arg Tyr LeuLys Val Asp Phe Ala 370 375 380 Asp Ile Gly Trp Asn Glu Trp Ile Ile SerPro Lys Ser Phe Asp Ala 385 390 395 400 Tyr Tyr Cys Ala Gly Ala Cys GluPhe Pro Met Pro Lys Ile Val Arg 405 410 415 Pro Ser Asn His Ala Thr IleGln Ser Ile Val Arg Ala Val Gly Ile 420 425 430 Val Pro Gly Ile Pro GluPro Cys Cys Val Pro Asp Lys Met Asn Ser 435 440 445 Leu Gly Val Leu PheLeu Asp Glu Asn Arg Asn Ala Val Leu Lys Val 450 455 460 Tyr Pro Asn MetSer Val Glu Thr Cys Ala Cys Arg 465 470 475 120 amino acids amino acidsingle linear protein GDF-10 Protein 1..120 6 Glu Lys Ser Met Gln LysAla Arg Arg Arg Gln Trp Asp Glu Pro Arg 1 5 10 15 Val Cys Ser Arg ArgTyr Leu Lys Val Asp Phe Ala Asp Ile Gly Trp 20 25 30 Asn Glu Trp Ile IleSer Pro Lys Ser Phe Asp Ala Tyr Tyr Cys Ala 35 40 45 Gly Ala Cys Glu PhePro Met Pro Lys Ile Val Arg Pro Ser Asn His 50 55 60 Ala Thr Ile Gln SerIle Val Arg Ala Val Gly Ile Val Pro Gly Ile 65 70 75 80 Pro Glu Pro CysCys Val Pro Asp Lys Met Asn Ser Leu Gly Val Leu 85 90 95 Phe Leu Asp GluAsn Arg Asn Ala Val Leu Lys Val Tyr Pro Asn Met 100 105 110 Ser Val GluThr Cys Ala Cys Arg 115 120 123 amino acids amino acid single linearprotein GDF-1 Protein 1..123 7 Arg Pro Arg Arg Asp Ala Glu Pro Val LeuGly Gly Gly Pro Gly Gly 1 5 10 15 Ala Cys Arg Ala Arg Arg Leu Tyr ValSer Phe Arg Glu Val Gly Trp 20 25 30 His Arg Trp Val Ile Ala Pro Arg GlyPhe Leu Ala Asn Tyr Cys Gln 35 40 45 Gly Gln Cys Ala Leu Pro Val Ala LeuSer Gly Ser Gly Gly Pro Prp 50 55 60 Ala Leu Asn His Ala Val Leu Arg AlaLeu Met His Ala Ala Ala Prp 65 70 75 80 Gly Ala Ala Asp Leu Pro Cys CysVal Pro Ala Arg Leu Ser Pro Ile 85 90 95 Ser Val Leu Phe Phe Asp Asn SerAsp Asn Val Val Leu Arg Gln Tyr 100 105 110 Glu Asp Met Val Val Asp GluCys Gly Cys Arg 115 120 118 amino acids amino acid single linear proteinGDF-3 Protein 1..118 8 Arg Lys Arg Arg Ala Ala Ile Ser Val Pro Lys GlyPhe Cys Arg Asn 1 5 10 15 Phe Cys His Arg His Gln Leu Phe Ile Asn PheGln Asp Leu Gly Trp 20 25 30 His Lys Trp Val Ile Ala Pro Lys Gly Phe MetAla Asn Tyr Cys His 35 40 45 Gly Glu Cys Pro Phe Ser Met Thr Thr Tyr LeuAsn Ser Ser Asn Tyr 50 55 60 Ala Phe Met Gln Ala Leu Met His Met Ala AspPro Lys Val Pro Lys 65 70 75 80 Ala Val Cys Val Pro Thr Lys Leu Ser ProIle Ser Met Leu Tyr Gln 85 90 95 Asp Ser Asp Lys Asn Val Ile Leu Arg HisTyr Glu Asp Met Val Val 100 105 110 Asp Glu Cys Gly Cys Gly 115 119amino acids amino acid single linear protein GDF-9 Protein 1..119 9 SerPhe Asn Leu Ser Glu Tyr Phe Lys Gln Phe Leu Phe Pro Gln Asn 1 5 10 15Glu Cys Glu Leu His Asp Phe Arg Leu Ser Phe Ser Gln Leu Lys Trp 20 25 30Asp Asn Trp Ile Val Ala Pro His Arg Tyr Asn Pro Arg Tyr Cys Lys 35 40 45Gly Asp Cys Pro Arg Ala Val Arg His Arg Tyr Gly Ser Pro Val His 50 55 60Thr Met Val Gln Asn Ile Ile Tyr Glu Lys Leu Asp Pro Ser Val Pro 65 70 7580 Arg Pro Ser Cys Val Pro Gly Lys Tyr Ser Pro Leu Ser Val Leu Thr 85 9095 Ile Glu Pro Asp Gly Ser Ile Ala Tyr Lys Glu Tyr Glu Asp Met Ile 100105 110 Ala Thr Arg Cys Thr Cys Arg 115 118 amino acids amino acidsingle linear protein BMP-2 Protein 1..118 10 Arg Glu Lys Arg Gln AlaLys His Lys Gln Arg Lys Arg Leu Lys Ser 1 5 10 15 Ser Cys Lys Arg HisPro Leu Tyr Val Asp Phe Ser Asp Val Gly Trp 20 25 30 Asn Asp Trp Ile ValAla Pro Pro Gly Tyr His Ala Phe Tyr Cys His 35 40 45 Gly Glu Cys Pro PhePro Leu Ala Asp His Leu Asn Ser Thr Asn His 50 55 60 Ala Ile Val Gln ThrLeu Val Asn Ser Val Asn Ser Lys Ile Pro Lys 65 70 75 80 Ala Cys Cys ValPro Thr Glu Leu Ser Ala Ile Ser Met Leu Tyr Leu 85 90 95 Asp Glu Asn GluLys Val Val Leu Lys Asn Tyr Gln Asp Met Val Val 100 105 110 Glu Gly CysGly Cys Arg 115 118 amino acids amino acid single linear protein BMP-4Protein 1..118 11 Lys Arg Ser Pro Lys His His Ser Gln Arg Ala Arg LysLys Asn Lys 1 5 10 15 Asn Cys Arg Arg His Ser Leu Tyr Val Asp Phe SerAsp Val Gly Trp 20 25 30 Asn Asp Trp Ile Val Ala Pro Pro Gly Tyr Gln AlaPhe Tyr Cys His 35 40 45 Gly Asp Cys Pro Phe Pro Leu Ala Asp His Leu AsnSer Thr Asn His 50 55 60 Ala Ile Val Gln Thr Leu Val Asn Ser Val Asn SerSer Ile Pro Lys 65 70 75 80 Ala Cys Cys Val Pro Thr Glu Leu Ser Ala IleSer Met Leu Tyr Leu 85 90 95 Asp Glu Tyr Asp Lys Val Val Leu Lys Asn TyrGln Glu Met Val Val 100 105 110 Glu Gly Cys Gly Cys Arg 115 119 aminoacids amino acid single linear protein Vgr-1 Protein 1..119 12 Ser ArgGly Ser Gly Ser Ser Asp Tyr Asn Gly Ser Glu Leu Lys Thr 1 5 10 15 AlaCys Lys Lys His Glu Leu Tyr Val Ser Phe Gln Asp Leu Gly Trp 20 25 30 GlnAsp Trp Ile Ile Ala Pro Lys Gly Tyr Ala Ala Asn Tyr Cys Asp 35 40 45 GlyGlu Cys Ser Phe Pro Leu Asn Ala His Met Asn Ala Thr Asn His 50 55 60 AlaIle Val Gln Thr Leu Val His Leu Met Asn Pro Glu Tyr Val Pro 65 70 75 80Lys Pro Cys Cys Ala Pro Thr Lys Leu Asn Ala Ile Ser Val Leu Tyr 85 90 95Phe Asp Asp Asn Ser Asn Val Ile Leu Lys Lys Tyr Arg Asn Met Val 100 105110 Val Arg Ala Cys Gly Cys His 115 119 amino acids amino acid singlelinear protein OP-1 Protein 1..119 13 Leu Arg Met Ala Asn Val Ala GluAsn Ser Ser Ser Asp Gln Arg Gln 1 5 10 15 Ala Cys Lys Lys His Glu LeuTyr Val Ser Phe Arg Asp Leu Gly Trp 20 25 30 Gln Asp Trp Ile Ile Ala ProGlu Gly Tyr Ala Ala Tyr Tyr Cys Glu 35 40 45 Gly Glu Cys Ala Phe Pro LeuAsn Ser Tyr Met Asn Ala Thr Asn His 50 55 60 Ala Ile Val Gln Thr Leu ValHis Phe Ile Asn Pro Glu Thr Val Pro 65 70 75 80 Lys Pro Cys Cys Ala ProThr Gln Leu Asn Ala Ile Ser Val Leu Tyr 85 90 95 Phe Asp Asp Ser Ser AsnVal Ile Leu Lys Lys Tyr Arg Asn Met Val 100 105 110 Val Arg Ala Cys GlyCys His 115 119 amino acids amino acid single linear protein BMP-5Protein 1..119 14 Ser Arg Met Ser Ser Val Gly Asp Tyr Asn Thr Ser GluGln Lys Gln 1 5 10 15 Ala Cys Lys Lys His Glu Leu Tyr Val Ser Phe ArgAsp Leu Gly Trp 20 25 30 Gln Asp Trp Ile Ile Ala Pro Glu Gly Tyr Ala AlaPhe Tyr Cys Asp 35 40 45 Gly Glu Cys Ser Phe Pro Leu Asn Ala His Met AsnAla Thr Asn His 50 55 60 Ala Ile Val Gln Thr Leu Val His Leu Met Phe ProAsp His Val Pro 65 70 75 80 Lys Pro Cys Cys Ala Pro Thr Lys Leu Asn AlaIle Ser Val Leu Tyr 85 90 95 Phe Asp Asp Ser Ser Asn Val Ile Leu Lys LysTyr Arg Asn Met Val 100 105 110 Val Arg Ser Cys Gly Cys His 115 119amino acids amino acid single linear protein OP-2 Protein 1..119 15 ArgLeu Pro Gly Ile Phe Asp Asp Val His Gly Ser His Gly Arg Gln 1 5 10 15Val Cys Arg Arg His Glu Leu Tyr Val Ser Phe Gln Asp Leu Gly Trp 20 25 30Leu Asp Trp Val Ile Ala Pro Gln Gly Tyr Ser Ala Tyr Tyr Cys Glu 35 40 45Gly Glu Cys Ser Phe Pro Leu Asp Ser Cys Met Asn Ala Thr Asn His 50 55 60Ala Ile Leu Gln Ser Leu Val His Leu Met Lys Pro Asn Ala Val Pro 65 70 7580 Lys Ala Cys Cys Ala Pro Thr Lys Leu Ser Ala Thr Ser Val Leu Tyr 85 9095 Tyr Asp Ser Ser Asn Asn Val Ile Leu Arg Lys Ala Arg Asn Met Val 100105 110 Val Lys Ala Cys Gly Cys His 115 120 amino acids amino acidsingle linear protein BMP-3 Protein 1..120 16 Glu Gln Thr Leu Lys LysAla Arg Arg Lys Gln Trp Ile Glu Pro Arg 1 5 10 15 Asn Cys Ala Arg ArgTyr Leu Lys Val Asp Phe Ala Asp Ile Gly Trp 20 25 30 Ser Glu Trp Ile IleSer Pro Lys Ser Phe Asp Ala Tyr Tyr Cys Ser 35 40 45 Gly Ala Cys Gln PhePro Met Pro Lys Ser Leu Lys Pro Ser Asn His 50 55 60 Ala Thr Ile Gln SerIle Val Arg Ala Val Gly Val Val Pro Gly Ile 65 70 75 80 Pro Glu Pro CysCys Val Pro Glu Lys Met Ser Ser Leu Ser Ile Leu 85 90 95 Phe Phe Asp GluAsn Lys Asn Val Val Leu Lys Val Tyr Pro Asn Met 100 105 110 Thr Val GluSer Cys Ala Cys Arg 115 120 116 amino acids amino acid single linearprotein MIS Protein 1..116 17 Gly Pro Gly Arg Ala Gln Arg Ser Ala GlyAla Thr Ala Ala Asp Gly 1 5 10 15 Pro Cys Ala Leu Arg Glu Leu Ser ValAsp Leu Arg Ala Glu Arg Ser 20 25 30 Val Leu Ile Pro Glu Thr Tyr Gln AlaAsn Asn Cys Gln Gly Val Cys 35 40 45 Gly Trp Pro Gln Ser Asp Arg Asn ProArg Tyr Gly Asn His Val Val 50 55 60 Leu Leu Leu Lys Met Gln Ala Arg GlyAla Ala Leu Ala Arg Pro Pro 65 70 75 80 Cys Cys Val Pro Thr Ala Tyr AlaGly Lys Leu Leu Ile Ser Leu Ser 85 90 95 Glu Glu Arg Ile Ser Ala His HisVal Pro Asn Met Val Ala Thr Glu 100 105 110 Cys Gly Cys Arg 115 122amino acids amino acid single linear protein Inhibin-alpha Protein1..122 18 Ala Leu Arg Leu Leu Gln Arg Pro Pro Glu Glu Pro Ala Ala HisAla 1 5 10 15 Asn Cys His Arg Val Ala Leu Asn Ile Ser Phe Gln Glu LeuGly Trp 20 25 30 Glu Arg Trp Ile Val Tyr Pro Pro Ser Phe Ile Phe His TyrCys His 35 40 45 Gly Gly Cys Gly Leu His Ile Pro Pro Asn Leu Ser Leu ProVal Pro 50 55 60 Gly Ala Pro Pro Thr Pro Ala Gln Pro Tyr Ser Leu Leu ProGly Ala 65 70 75 80 Gln Pro Cys Cys Ala Ala Leu Pro Gly Thr Met Arg ProLeu His Val 85 90 95 Arg Thr Thr Ser Asp Gly Gly Tyr Ser Phe Lys Tyr GluThr Val Pro 100 105 110 Asn Leu Leu Thr Gln His Cys Ala Cys Ile 115 120121 amino acids amino acid single linear protein Inhibin-beta-A Protein1..121 19 Arg Arg Arg Arg Arg Gly Leu Glu Cys Asp Gly Lys Val Asn IleCys 1 5 10 15 Cys Lys Lys Gln Phe Phe Val Ser Phe Lys Asp Ile Gly TrpAsn Asp 20 25 30 Trp Ile Ile Ala Pro Ser Gly Tyr His Ala Asn Tyr Cys GluGly Glu 35 40 45 Cys Pro Ser His Ile Ala Gly Thr Ser Gly Ser Ser Leu SerPhe His 50 55 60 Ser Thr Val Ile Asn His Tyr Arg Met Arg Gly His Ser ProPhe Ala 65 70 75 80 Asn Leu Lys Ser Cys Cys Val Pro Thr Lys Leu Arg ProMet Ser Met 85 90 95 Leu Tyr Tyr Asp Asp Gly Gln Asn Ile Ile Lys Lys AspIle Gln Asn 100 105 110 Met Ile Val Glu Glu Cys Gly Cys Ser 115 120 120amino acids amino acid single linear protein Inhibin-beta-B Protein1..120 20 Arg Ile Arg Lys Arg Gly Leu Glu Cys Asp Gly Arg Thr Asn LeuCys 1 5 10 15 Cys Arg Gln Gln Phe Phe Ile Asp Phe Arg Leu Ile Gly TrpAsn Asp 20 25 30 Trp Ile Ile Ala Pro Thr Gly Tyr Tyr Gly Asn Tyr Cys GluGly Ser 35 40 45 Cys Pro Ala Tyr Leu Ala Gly Val Pro Gly Ser Ala Ser SerPhe His 50 55 60 Thr Ala Val Val Asn Gln Tyr Arg Met Arg Gly Leu Asn ProGly Thr 65 70 75 80 Val Asn Ser Cys Cys Ile Pro Thr Lys Leu Ser Thr MetSer Met Leu 85 90 95 Tyr Phe Asp Asp Glu Tyr Asn Ile Val Lys Arg Asp ValPro Asn Met 100 105 110 Ile Val Glu Glu Cys Gly Cys Ala 115 120 118amino acids amino acid single linear protein Nodal Protein 1..118 21 GlyTrp Gly Arg Arg Gln Arg Arg His His Leu Pro Asp Arg Ser Gln 1 5 10 15Leu Cys Arg Arg Val Lys Phe Gln Val Asp Phe Asn Leu Ile Gly Trp 20 25 30Gly Ser Trp Ile Ile Tyr Pro Lys Gln Tyr Asn Ala Tyr Arg Cys Glu 35 40 45Gly Glu Cys Pro Asn Pro Val Gly Glu Glu Phe His Pro Thr Asn His 50 55 60Ala Tyr Ile Gln Ser Leu Leu Lys Arg Tyr Gln Pro His Arg Val Pro 65 70 7580 Ser Thr Cys Cys Ala Pro Val Lys Thr Lys Pro Leu Ser Met Leu Tyr 85 9095 Val Asp Asn Gly Arg Val Leu Leu Glu His His Lys Asp Met Ile Val 100105 110 Glu Glu Cys Gly Cys Leu 115 114 amino acids amino acid singlelinear protein TGF-beta-1 Protein 1..114 22 Arg Arg Ala Leu Asp Thr AsnTyr Cys Phe Ser Ser Thr Glu Lys Asn 1 5 10 15 Cys Cys Val Arg Gln LeuTyr Ile Asp Phe Arg Lys Asp Leu Gly Trp 20 25 30 Lys Trp Ile His Glu ProLys Gly Tyr His Ala Asn Phe Cys Leu Gly 35 40 45 Pro Cys Pro Tyr Ile TrpSer Leu Asp Thr Gln Tyr Ser Lys Val Leu 50 55 60 Ala Leu Tyr Asn Gln HisAsn Pro Gly Ala Ser Ala Ala Pro Cys Cys 65 70 75 80 Val Pro Gln Ala LeuGlu Pro Leu Pro Ile Val Tyr Tyr Val Gly Arg 85 90 95 Lys Pro Lys Val GluGln Leu Ser Asn Met Ile Val Arg Ser Cys Lys 100 105 110 Cys Ser 114amino acids amino acid single linear protein TGF-beta-2 Protein 1..11423 Lys Arg Ala Leu Asp Ala Ala Tyr Cys Phe Arg Asn Val Gln Asp Asn 1 510 15 Cys Cys Leu Arg Pro Leu Tyr Ile Asp Phe Lys Arg Asp Leu Gly Trp 2025 30 Lys Trp Ile His Glu Pro Lys Gly Tyr Asn Ala Asn Phe Cys Ala Gly 3540 45 Ala Cys Pro Tyr Leu Trp Ser Ser Asp Thr Gln His Ser Arg Val Leu 5055 60 Ser Leu Tyr Asn Thr Ile Asn Pro Glu Ala Ser Ala Ser Pro Cys Cys 6570 75 80 Val Ser Gln Asp Leu Glu Pro Leu Thr Ile Leu Tyr Tyr Ile Gly Lys85 90 95 Thr Pro Lys Ile Glu Gln Leu Ser Asn Met Ile Val Lys Ser Cys Lys100 105 110 Cys Ser 114 amino acids amino acid single linear proteinTGF-beta-3 Protein 1..114 24 Lys Arg Ala Leu Asp Thr Asn Tyr Cys Phe ArgAsn Leu Glu Glu Asn 1 5 10 15 Cys Cys Val Arg Pro Leu Tyr Ile Asp PheArg Gln Asp Leu Gly Trp 20 25 30 Lys Trp Val His Glu Pro Lys Gly Tyr TyrAla Asn Phe Cys Ser Gly 35 40 45 Pro Cys Pro Tyr Leu Arg Ser Ala Asp ThrThr His Ser Thr Val Leu 50 55 60 Gly Leu Tyr Asn Thr Leu Asn Pro Glu AlaSer Ala Ser Pro Cys Cys 65 70 75 80 Val Pro Gln Asp Leu Glu Pro Leu ThrIle Leu Tyr Tyr Val Gly Arg 85 90 95 Thr Pro Lys Val Glu Gln Leu Ser AsnMet Val Val Lys Ser Cys Lys 100 105 110 Cys Ser 115 amino acids aminoacid single linear protein Human GDF-10 Protein 1..115 25 Lys Ala ArgArg Lys Gln Trp Asp Glu Pro Arg Val Cys Ser Arg Arg 1 5 10 15 Tyr LeuLys Val Asp Phe Ala Asp Ile Gly Trp Asn Glu Trp Ile Ile 20 25 30 Ser ProLys Ser Phe Asp Ala Tyr Tyr Cys Ala Gly Ala Cys Glu Phe 35 40 45 Pro MetPro Lys Ile Val Arg Pro Ser Asn His Ala Thr Ile Gln Ser 50 55 60 Ile ValArg Ala Val Gly Ile Ile Pro Gly Ile Pro Glu Pro Cys Cys 65 70 75 80 ValPro Asp Lys Met Asn Ser Leu Gly Val Leu Phe Leu Asp Glu Asn 85 90 95 ArgAsn Val Val Leu Lys Val Tyr Pro Asn Met Ser Val Asp Thr Cys 100 105 110Ala Cys Arg 115 115 amino acids amino acid single linear protein MurineGDF-10 Protein 1..115 26 Lys Ala Arg Arg Lys Gln Trp Asp Glu Pro Arg ValCys Ser Arg Arg 1 5 10 15 Tyr Leu Lys Val Asp Phe Ala Asp Ile Gly TrpAsn Glu Trp Ile Ile 20 25 30 Ser Pro Lys Ser Phe Asp Ala Tyr Tyr Cys AlaGly Ala Cys Glu Phe 35 40 45 Pro Met Pro Lys Ile Val Arg Pro Ser Asn HisAla Thr Ile Gln Ser 50 55 60 Ile Val Arg Ala Val Gly Ile Val Pro Gly IlePro Glu Pro Cys Cys 65 70 75 80 Val Pro Asp Lys Met Asn Ser Leu Gly ValLeu Phe Leu Asp Glu Asn 85 90 95 Arg Asn Ala Val Leu Lys Val Tyr Pro AsnMet Ser Val Glu Thr Cys 100 105 110 Ala Cys Arg 115

We claim:
 1. A purified antibody that binds to growth differentiationfactor-10 (GDF-10) or to immuogenic fragments thereof.
 2. The antibodyof claim 1, wherein the antibody is polyclonal.
 3. The antibody of claim1, wherein the antibody is monoclonal.
 4. The antibody of claim 1,wherein the antibody includes Fab, Fv or other functional antibodyfragments.